1. Field of the Invention
This invention pertains to the field of diagnostic assays for detecting infection of an animal by the protozoan parasite Entamoeba histolytica.
2. Background
Entamoeba histolytica affects an estimated 480 million people annually; about 10 percent of these people develop colitis, liver abscesses, or other symptoms. Recently, a non-pathogenic species, E. dispar, has been described (Diamond and Clark (1993) J. Euk. Microbiol. 40: 340-344). E. dispar is morphologically identical to the pathogenic species E. histolytica.
Diagnosis of E. histolytica infection is often difficult. Amoebic dysentery caused by E. histolytica is easily confused with monocytic erythrophagocytosis and erythrophagocytosis caused by Entamoeba coli (Long and Christie (1995) Clin. Lab. Med. 15: 307-331). Early diagnostic assays included microscopy and culture. One more recently developed diagnostic method involves detection of Entamoeba-specific IgG, IgM and IgA antibodies in serum (Healy (1986) Rev. Infect. Dis. 8: 239-246; Arvind et al. (1988) Serodiagn. Inmunother. Infect. Dis. 2: 79-84). However, seropositivity can persist for years, thus resulting in a high background due to healthy subjects giving positive results (Krupp (1970) Am. J. Trop. Med. Hyg. 19: 57-62; Lobel et al. (1970) Ann. Rev. Microbiol. 32: 379-347).
Another diagnostic method involves detection of a lectin found on the surface of E. histolytica and E. dispar trophozoites. Infection of a cell by Entamoeba involves binding of this lectin to Gal/Ga1NAc residues on the surface of the target cell (Petri et al. (1989) J. Biol. Clem. 264: 3007-3012). The lectin, which has a molecular mass of 260 kDa, is composed of two subunits of 170 kDa and 35 kDa. Diagnostic assays that use monoclonal antibodies raised against purified native 170 kDa antigen were found to have problems with false positives (Ravdin et al. (1990) J. Infect. Dis. 162: 768-772). Monoclonal antibodies against a recombinantly produced form of the 170 kDa subunit and the use of the antibodies for detecting the 170 kDa antigen are discussed in U.S. Pat. No. 5,272,058 (see also, Mann et al. (1993) Infect. Immun. 61: 1772-1778; Petri et al. (1990) Infect. Immun. 58: 1802-1806). Other immunoassays for diagnosing E. histolytica infection are discussed in, for example, Root et al. (1978) Arch. Invest. Med. (Mex) 9: Supplement 1: 203.
Two commercially available immunoassays for E. histolytica detection were recently compared to culture and PCR methods (Haque et al. (1996) 96.sup.th ASM General Meeting, New Orleans La.). The TechLab "Entamoeba Test" uses a monoclonal antibody to detect a the 170 kDa subunit of the Gal/Ga1NAc lectin that is present in both pathogenic E. histolytica and non-pathogenic E. dispar. The Alexon "ProSpecT Entamoeba histolytica Microplate Assay" also detects both pathogenic and non-pathogenic Entamoeba species, through use of rabbit polyclonal antisera. Comparison of these two tests to PCR and/or culture methods found that the Alexon test had a sensitivity of only 55% and a correlation of 66%, while the TechLab test had a sensitivity of 100% and a correlation of 84% (Id.). Both tests, however, are unable to distinguish between pathogenic and non-pathogenic strains.
Pathogenic E. histolytica trophozoites display a 29 kDa cysteine-rich surface antigen (Torian et al. (1990) Proc. Nat'l Acad. Sci. USA 87: 6358-6362). Monoclonal antibodies raised against this antigen were tested for ability to detect E. histolytica infection, but not all clinical isolates were detected (Id.). Thus, a need exists for sensitive and reliable assays for detecting E. histolytica infection in a clinical setting. The present invention fulfills this and other needs.